Regulation of GABA_A receptors by fragile X mental retardation protein

Abstract

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP). The deficiency of GABA_A receptors (GABA_ARs) is implicated in FXS. However, the underlying mechanisms remain unclear. To investigate the effect of FMRP on GABA_ARs, we transfected FMRP cDNAs in rat cortical neurons. We measured the protein expression of GABA_ARs and phosphatase PTEN, and recorded GABA_AR-mediated whole-cell currents in the transfected neurons. We show that the transfection of FMRP cDNAs causes increased protein expression of GABA_ARs in cortical neurons, but GABA_AR-mediated whole-cell currents are not potentiated by FMRP transfection. These results suggest the possibility that intracellular signaling antagonizing GABA_AR activity may play a role in inhibiting GABA_AR function in FMRP-transfected neurons. We further show that FMRP transfection results in an enhanced protein expression of PTEN, which contributes to the inhibition of GABA_AR function in FMRP-transfected neurons. These results indicate that GABA_ARs are regulated by FMRP through both an up-regulation of GABA_AR expression and a PTEN enhancement-induced inhibition of GABA_AR function, suggesting that an abnormal regulation of GABA_AR and PTEN by the loss of FMRP underlies the pathogenesis of FXS.

Introduction

Fragile X syndrome (FXS), caused by the loss of fragile X mental retardation protein (FMRP), is the most common inherited form of mental retardation [1-3]. The trinucleotide CGG expansion that inactivates the fragile X mental retardation 1 (FMR1) gene prevents the expression of the encoded FMRP protein [4]. FMRP is a selective RNA-binding protein that regulates the local translation of a subset of mRNAs at synapses [5]. The major symptoms of FXS are mental retardation, autistic behaviors, attention deficit, hyperactivity, alteration in sleep patterns and epileptic seizures [6,7].

The γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS) [8]. The GABA subtype A receptors (GABA_ARs) are generally localized on the postsynaptic membranes and responsible for most fast inhibitory synaptic transmission in the CNS through opening bicuculline-sensitive Cl^- channels [9]. GABA_ARs are assembled from several different classes of subunits (α1-6, β1-3, γ1-3, δ, θ, π and ε) and the α1β2γ2 combination is the most abundant GABA_ARs expressed in the brain [10,11]. Recent evidence indicates that the mRNA levels of seven subunits of GABA_AR, including α1, α3, α4, β1, β2, γ1 and γ2, are decreased in the cortex of FMR1 knockout mice [12]. The protein level of GABA_AR β subunit is also reduced in cortex, hippocampus, diencephalons and brainstem of fragile X mice [13]. Electrophysiological studies suggest that the GABAergic efficiency and the tonic GABA_AR currents may be suppressed in the fragile X mice [14-16]. Moreover, anatomical defects have been observed in the neocortical GABAergic inhibitory circuits [16]. In agreement with the alterations of GABA_ARs, the ratio between inhibitory (taurine and GABA) and excitatory (aspartate and glutamate) amino acids is decreased in brainstem, hippocampus and caudal cortex of fragile X mouse [17]. These findings suggest that the absence of FMRP may be involved in mediating the suppressed activities of GABA_ARs in FXS. As dysfunction of GABA_AR channels is implicated in symptoms that are also disturbed in fragile X patients, such as anxiety, depression, epilepsy, insomnia, and learning and memory [18], it is likely that the decreased GABA_AR function may underlying the behavioral and epileptic phenotype associated with FXS [19].

PTEN (Phosphatase and tensin homolog deleted on chromosome ten) is a dual-specificity phosphatase [20]. Recently, we have provided evidence that PTEN can positively regulate both the expression and function of excitatory NMDA receptors in rat hippocampal neurons [21,22]. Suppressing PTEN protects ischemia-induced neuronal death through both inhibiting NMDA receptor-mediated excitotoxicity and enhancing activity of cell survival-promoting kinase Akt [21,22]. We also showed that PTEN negatively regulates GABA_AR function in hippocampal neurons [23].

To reveal the pathogenesis of FXS, a necessary step is to understand the biological role of FMRP in the CNS. We therefore set up to test the interactions among FMRP, GABA_AR and PTEN in an experimental model with FMRP overexpression in cultured rat cortical neurons.

Materials and methods

Cortex neuronal culture

Cortex neuronal cultures were prepared from Wistar rats on gestation day 18 [24]. Dissociated neurons were suspended in plating medium (Neurobasal medium, 2% B-27 supplement, 0.5% FBS, 0.5 μM L-glutamine, and 25 μM glutamic acid) and transferred to poly-D-lysine-coated coverslips in 35mm Petri dishes. After 3 d in vitro (DIV), half of the plating medium was removed and replaced with maintenance medium (Neurobasal medium, B-27 supplement, and 0.5 μM L-glutamine). Medium replacement was performed every 3-4 d, and cells were used at 12-15 DIV.

Immunofluorescent labeling, image acquisition and analysis

To examine the surface expression of GABA_AR γ2 subunits, nonpermeabilized cells were labeled with rabbit anti-GABA_AR γ2 antibody (Millipore Corporation, Billerica, MA), and Alexa Fluor 594 (red fluorescence) secondary antibody (Invitrogen, Burlington, Ontario, Canada). The detailed methods of surface receptor labeling are described in our previous studies [25]. To examine FMRP or PTEN expression, cells were permeabilized with 4% paraformaldehyde/PBS and 0.3% Triton X-100, and then labeled with rabbit anti-PTEN antibody (Cell Signaling Technology, Inc. Danvers, MA) or rabbit anti-FMRP polyclonal antibody (Abcam, Cambridge, MA).

Fluorescence-labeled neurons were imaged using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Germany) and analyzed as described previously [25-28]. Images were acquired using a Zeiss AxioCam digital camera in the linear range with constant settings. Each image was a z-series of 6-13 images, taken at 0.75-μm-depth intervals. The resultant stack was “flattened” into a single image using a maximum projection. For all experiments, we analyzed fluorescent signal in regions of interest by measuring the average fluorescence intensity per unit area. All images in all experiments were analyzed using identical acquisition parameters. During data acquisition and analysis, the investigator was blind to the treatment group. In each experiment, neurons were selected randomly under bright-field optics, and fluorescent images of each neuron acquired from a single plane were transferred for analysis. The cells in control and OGD groups from the same culture preparation were processed and imaged in parallel. Three fields were randomly selected in each culture. The fluorescence density was analyzed by Image J software (NIH) [25,29,30]. All the immunolabeling experiments were repeated using neuronal cultures prepared from 5-8 animals. The expression of surface receptors and whole-cell proteins represented by labeled fluorescence densities in treated groups was normalized versus that in control groups. The n value refers to the number of cells analyzed.

Transfection

The transfection of GFP (green fluorescence protein) cDNA, wild-type FMRP-GFP (FMRP-GFP) cDNA, scrambled PTEN siRNA (SsiRNA-pten) or PTEN siRNA (siRNApten) in cultured cortical neurons was done using Lipofectamine 2000 (Invitrogen) as described previously [31],GFP positive cells were selected for immunostaining analysis.

Recording of GABA_AR-mediated whole cell currents

Whole-cell patch-clamp recording was performed as described previously [21,25]. The recording electrode was filled with solution containing 140 mM CsCl, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM EGTA, 10 mM HEPES, 4 mM K2ATP, with pH=7.3, osmolality=280-290 mOsm and resistance=3-5 MΏ. The extracellular solution contains (in mM): 140 NaCl, 2.0 CaCl₂, 1.0 MgCl₂, 5.0 KCl, 25 HEPES, 33 glucose (pH 7.35, osmolarity 320 mOsm/L). BpV(pic) (CalBiochem, EMD Chemicals, Inc. San Diego, CA) was added into the pipette filling solution. TTX (0.5 μM) was added into the bath solution to block voltage- gated sodium channels. Neurons were held at -60 mV under voltage clamp. GABA_AR-mediated whole-cell currents were recorded by pressure application of 100 μM GABA (20 kPa, 20 ms) from a micropipette with its tip located -20 μm from the recorded cell. GABA were delivered at intervals of 30 s. Data were acquired with an Axopatch 200B amplifier and pClamp 10 software interfaced to a Digidata 1322A acquisition board (Molecular Devices, CA), and signals were filtered at 2 kHz and digitized at 10 kHz.

Statistics

Student’s t test or ANOVA test was used where appropriate to examine the statistical significance of the differences between groups of data. Significance was placed at p<0.05.

Results

FMRP enhances the surface expression of GABA_ARs

To determine whether FMRP regulates GABA_ARs, we examined the membrane expression of GABA_ARs in FMRP-overexpressed cortical neurons. A non-permeable staining method was used to examine the surface expression of GABA_AR γ2 subunits in the cultured cortical neurons transfected with wild-type FMRP cDNAs that was conjugated with GFP [25]. As illustrated in Figure 1A, neurons transfected with FMRP-GFP exhibit increased expression of FMRP protein. Using a polyclonal antibody against the extracellular N-terminus of GABA_AR γ2 subunit, we showed that the surface expression of γ2 subunits was significantly increased in neurons transfected with FMRP-GFP, compared with that in neurons transfected with GFP alone (Figure 1B). These data indicate that FMRP can positively regulate the protein expression of GABA_AR γ2 subunits in the membrane surface of cortical neurons.

Figure 1.

The surface expression of GABA_AR γ2 subunits is increased by FMRP upregulation. A: Immunofluorescent staining of FMRP (red) in neurons transfected with cDNAs of GFP and wild-type FMRP-GFP, respectively. Summarized data show that the expression of FMRP is increased in cultured cortical neurons transfected with FMRP-GFP (n=7 for both groups, *p<0.05, Student’s t test). B: Non-permeable immunofluorescent staining of membrane surface GABA_AR γ2 subunits (red) in neurons transfected with cDNAs of GFP and FMRP-GFP, respectively. Summarized data indicate that FMRP upregulation increases γ2 surface expression in cortical neurons transfected with cDNAs of FMRP-GFP (n=6 for both groups, *p<0.05, Student’s t test).

FMRP does not alter the function of GABA_ARs

As the increased surface GABA_AR expression might contribute to an enhanced function of these channels, we recorded GABA_AR-mediated whole-cell currents in cultured cortical neurons transfected with FMRP-GFP or GFP alone. Surprisingly, our results showed that GABA_AR-mediated currents were not significantly increased in neurons transfected with FMRP-GFP compared with neurons transfected with GFP alone (Figure 2). Among many possibilities, a simple explanation for this result is that the FMRP up-regulation of GABA_AR function may be antagonized by intracellular signaling that are also regulated by FMRP.

Figure 2.

The GABA_AR-mediated whole-cell currents are not altered by FMRP upregulation. Left, sample traces of GABA_AR-mediated whole-cell currents recorded in neurons transfected with GFP and FMRP-GFP, respectively. Right, the summarized data show that FMRP does not alter GABA_AR-mediated whole-cell currents (n=6 for GFP group, n=8 for FMRP-GFP group; *p<0.05, Student's t test).

FMRP increases PTEN expression

Our recent study shows that the phosphatase PTEN negatively regulates GABA_ARs in rat hippocampal neurons [23]. We therefore hypothesized that the enhancement of GABA_AR function by FMRP may be suppressed by the increased PTEN expression in FMRP-over-expressed neurons. Indeed, our results showed that FMRP overexpression significantly increased protein expression of PTEN in cultured neurons(Figure 3). These data suggest that the increased PTEN expression in FMRP-over-expressed neurons may inhibit GABA_AR function.

Figure 3.

FMRP overexpression enhances PTEN expression in cortical neurons. Left, representative images showing immunofluorescent staining of PTEN (red) in neurons transfected with cDNAs of GFP and FMRP-GFP, respectively. Right, summarized data show that PTEN expression is increased in FMRP-overexpressed neurons (n=7 for GFP group, n=6 for FMRP-GFP group; p<0.05, Student's t test).

FMRP suppresses GABA_AR function through upregulation of PTEN

To determine whether an increased PTEN expression in FMRP-overexpressed cortical neurons could inhibit GABA_AR function, we tested the effects of PTEN inhibitor BpV(pic) on GABA_AR-mediated whole-cell currents in cultured neurons transfected with cDNAs of GFP or FMRP-GFP [32,33]. Our data showed that PTEN inhibition by BpV(pic) significantly increased the peak currents of GABA_ARs in neurons transfected with FMRP-GFP (Figure 4A), suggesting that the upregulation of endogenous PTEN by FMRP inhibits GABA_AR function in cortical neurons. Thus, the elevated PTEN expression counteracts the effect of FMRP-induced increase of GABA_AR expression. To provide evidence that BpV(pic) is a specific PTEN inhibitor in rat cortical neurons, neurons transfected with scrambled PTEN siRNA (SsiRNApten) or PTEN siRNA (siRNApten) were treated with BpV(pic). We showed that while SsiRNApten had no effect on BpV(pic)-induced potentiation of GABA_AR currents, siRNApten introduction occluded BpV(pic)-induced potentiation of GABA_AR currents (Figure 4B-D), indicating the specificity of BpV(pic) in inhibiting PTEN activity in our experimental conditions. Taken together, this study reveals that GABA_ARs are regulated by FMRP through both an up-regulation of GABA_AR expression and a PTEN enhancement-induced inhibition of GABA_AR function.

Figure 4.

FMRP inhibits GABA_AR function through upregulation of PTEN. A: Sample traces of GABA_AR-mediated whole-cell currents recorded in neurons transfected with GFP and FMRP-GFP, respectively. Summarized data show that PTEN inhibition by bpV(pic) increases the peak amplitude of GABA_AR currents in neurons transfected with FMRP-GFP (n=7 for GFP group, n=8 for FMRP-GFP group; *p<0.05, Student’s t test). B & D: SsiRNApten transfectionhas no effect on bpV(pic)-induced potentiation of GABA_AR currents (n=7 for GFP group, n=7 for FMRP-GFP group; *p<0.05, ANOVA test). C & D: siRNApten introduction occludes bpV(pic)-induced potentiation of GABA_AR currents (n=8 for GFP group, n=8 for FMRP-GFP group; *p<0.05, ANOVA test).

Discussion

Epileptic seizure is a disorder of recurrent, spontaneous episodes of aberrant synchronization in neural networks [34]. It has been reported that about 10-20% of FXS patients suffered from seizures [35]. Increasing evidence suggests that GABA_AR deficiency may contribute to the occurrence of epileptic seizures in FXS [35]. Based on our experimental evidence obtained from the FMRP overexpression model, we reason that the absence of FMRP in FXS may lead to reduced protein expression of both GABA_ARs and PTEN. As PTEN suppression can potentiate GABA_AR function, the effect of suppressed GABA_AR expression in FMRP-deficient neurons that is supposed to cause increased seizure occurrence, would be antagonized by FMRP loss-induced PTEN suppression. If this is true, the PTEN inhibition-mediated GABA_AR upregulation may explain in part why only 10-20% of fragile X patients have seizure occurrence [35].

Our previous study demonstrates that PTEN increases NMDA receptor activity by physically associating with NR2B-containing NMDA receptors [21,22]. It is possible that FMRP loss-induced PTEN suppression may also actthrough inhibiting NMDA receptor-m-ediated excitatory activity to counte-ract seizure occurrencein FXS.

Yet, it is unclear how PTEN exerts its effect on GABA_ARs. Our future studies will investigate whether PT-EN regulates GABA_AR function through a direct protein-protein interaction as PTEN regulation of NM-DA receptors [21]. If not, intracellular signaling mediates PTEN regulation of GABA_ARs will be investigated.We will also investigate in detail whether the channel properties of GABA_ARs and the GABA_AR-medi-ated synaptic responses are regulated by FMRP.

In summary, the present study provides evidence that FMRP and PT-EN play opposite roles in regulating GABA_ARs in cortical neurons. While FMRP enhances GABA_AR expression, it also increases the protein expression of PT-EN, which in turn antagonizes FMRP-induced potentiation of GABA_ARs. These results suggest that PTEN downregulation may play a protective role in reducing GABA_AR deficiency-induced incidence of epileptic seizures in FXS.